Pathogens don't carry passports Taxonomic aspects of the Psa story

Bevan Weir Landcare Research, Auckland Landcare Research Link Seminar, Wellington, 28 May 2013 capability

• With Landcare Research for 11 years – PhD 2006: rhizobia – Postdoc 2011: Colletotrichum • Fungi & team: 5 scientists, 5 technicians • In Auckland – Co-located with MPI PHEL

Outline

• Pathogens don’t carry passports – How do we know what they are? • How taxonomy + collections + databases can help inform policy decisions • With reference to the recent Psa kiwifruit disease – But relevant to many plant pathogens and fungi – PTA, Myrtle rust, Citrus canker, Xylella etc. What is Psa?

• A bacterial disease of kiwifruit – Leaf spots, canker, death • syringae pv. actinidiae – Trinomial name? – bacterium

Severe kiwifruit disease

• Psa-V Statistics – 22 May 2013 • 2102 orchards have Psa-V. • 71% of New Zealand’s kiwifruit hectares • Not Nelson

kvh.org.nz Why Landcare Research?

• MPI – Initial incursion response – Regulations • Plant and Food Research (PFR) – Industry links – Developed kiwifruit cultivars (Royalties) – Plant pathologists • KVH – Independent org. managing Psa Why Landcare Research?

• History • DSIR split into CRIs in 1992 • The PDD of DSIR was split between Crop & Food, Hort Research, AgResearch, and Landcare Research • Landcare retained: – National collections (CHR, PDD, NZAC, ICMP) – Taxonomists DSIR Bacteriology 1988 Psa response Genome sequence

• November 2010 from Te Puke • MAF ID’d bacterium as Psa • But which strain? – Asian – Italian • Asked by MAF to do whole genome sequence – Sequenced in 3 days – Capability from in-house research tool (454) – Microsatellite discovery Genome analysis Gene Japan 84, Italy Italy 08/09 NZ 2010 94 avrPto1 - - avrD1 + + avrAE1 + + hopA1 - + hopB1 - - hopC1 - - hopD1 + + hopF2 - - hopG1 - - hrpK1 + + hopAF1 ± - hopAN1 + + Coronatine ± - Phaseolotoxin + - Genome analysis Gene Japan 84, Italy Italy 08/09 NZ 2010 94 avrPto1 - - - avrD1 + + + avrAE1 + + + hopA1 - + + hopB1 - - - hopC1 - - - hopD1 + + + hopF2 - - - hopG1 - - - hrpK1 + + + hopAF1 ± - - hopAN1 + + + Coronatine ± - - Phaseolotoxin + - - ICMP: International Collection of Microorganisms from plants • National coll. of living bacteria and fungi – All of NZ, stakeholders

– Liquid N2 • 18,800 cultures • Important collection of plant pathogenic bacteria – “type strain” of Psa (Takikawa 1989) New or Old disease in NZ?

• Was Psa-V a recent introduction? • Or been here for a long time – changing conditions caused severe disease? • Screened 40 years of ex- kiwifruit bacteria in the ICMP looking for Psa – Found no matches from 143 • Value of collections to NZ – Keep collecting NZ material Taxonomy • V. important group of plant pathogens – 11 species and 64 pathovars (26 in NZ) – Pathovar = Pathological variants – Inconsistent confusing taxonomy – 7 “Genomospecies” • Pseudomonas syringae pv. actinidiae • Bacteriological Code & Pathovar Standards – Rules of nomenclature – pv. system developed in NZ P. syringae complex

Pseudomonas syringae pv. syringae - Lilac Bacterial blight P. syringae complex

Pseudomonas savastanoi pv. savastanoi - Olive knot disease P. syringae complex

Pseudomonas syringae pv. helianthi - Sunflower leaf spot P. syringae complex

Pseudomonas syringae pv. coronafaciens - Oat halo blight P. syringae complex

Pseudomonas cannabina pv. cannabina - leaf and stem rot P. syringae complex

Pseudomonas syringae pv. actinidiae - Kiwifruit bacterial canker P. syringae complex

P. syringae “bud rot”

P. syringae pv. syringae

Pseudomonas syringae pv. actinidiae - Kiwifruit bacterial canker

P. marginalis Pseudomonas syringae Taxonomy Research • Multigene sequencing – Novel markers derived from genome sequences • Taxonomic revision: Will split into 7 species – e.g. P. avellanae pv. actinidiae – Consistent species concept

Different Psa populations in NZ?

• The genome matched ‘Italian’ • Diagnostic test of the time did not differentiate populations – Testing from around the country indicated Psa was widespread – Impacted policy & biosecurity decisions • But subsequent sequencing revealed a novel Psa population in NZ – Psa-V: strong pathogen – Psa-LV: Leaf spots, plants survive, been here a while Psa global populations

I-Psa Z Italy (2008) I-Psa 8 Italy (2008) CRA-FRU 10.22 Italy (2008) CFPB 7286 Italy (2008) CRA-FRU 8.43 Italy (2008) CRA-FRU 8.76 Italy (2009) CRA-FRU 11.40 Italy (2010) CRA-FRU 11.41 Italy (2010) CRA-FRU 11.42 Italy (2010) CRA-FRU 11.43 Italy (2010) I.27.4.10.D1 Italy (2010) 0.91 I.6.5.10.4-4 Italy (2010) T10_04758 New Zealand (2010) Psa3 T10_04782 New Zealand (2010) T10_05815 New Zealand (2010) T10_05454 New Zealand (2010) (Psa-V) T10_06115 New Zealand (2010) T11_0918 New Zealand (2011) CH2010-6 China (2010) 1 Psa1A Chile (2010) Psa1B Chile (2010) 286532 Chile (2010) 2871B5 Chile (2010) 287A63 Chile (2010) 1 ICMP 9855 Japan (1984) MAFF 302143 Japan (1987) 1 NCPPB 3739 Japan (1984) PT 1 MAFF 302091 Japan (1984) MAFF 302145 Japan (1988) Psa1 PA 459 Japan (1988) NCPPB 3871 Italy (1994) NCPPB 3873 Italy (1994) KACC 10584 Korea (1997) 1 KACC 10594 Korea (1998) KACC 10754 Korea (1997) Psa2 T10_04976 New Zealand (2010) T10_05163 New Zealand (2010) T10_05188 New Zealand (2010) T10_05195 New Zealand (2010) 1 T10_05196 New Zealand (2010) Psa4 T11_01581 Australia (1990) T11_01369A Australia (2011) (Psa-LV) T11_01395A Australia (2011) 28830 Australia (2011)

Chapman et al. 2012 – doi:10.1094/PHYTO-03-12-0064-R Regulation on species?

• Taxonomic hierarchy – Species (P. syringae) – Pathovars (pv. actinidiae) – Populations (Psa3) • Which level to regulate at? • Pathogenicity genes? – Psa3 (Psa-V) and Psa4 (Psa-LV) present in NZ – Psa1 and Psa2 absent but less virulent – Introduction may introduce new path genes Regulation on species?

• Do pre-emptive work on emerging pathogens – If we knew what we know now back in 2010 different decisions could have been made – e.g myrtle rust – Collb. With B3 / national sci challenges • Taxonomist provide sensible species concepts • Investigate population level differences Diagnostics

Psa diagnostic service

• CRIs have immediate capability • Did 300+ Psa tests by isolation + seq. – Living bacteria gold standard – Into the ICMP • Trained commercial labs – Tech transfer • Now occasional tests • Did sequencing for MAF – weekends New diagnostic tests

• Helped validate new diagnostic tests – Faster, more specific – Directly from plant tissue • Provided positive controls and 150+ DNA – genetically similar pathovars – other ex-kiwifruit bacteria – Based on P. syringae taxonomy research • Validation very important – False positives have economic consequences New plant pathogen facility

• “PC2+” containment – HEPA filter – Steam water waste – Shower out • Unique capability • Enabling new research – Testing pathogens and biocontol agents in NZ • PFR using facility for Psa research NZfungi database

• Database of Fungi and bacteria – Pansectorial, national database – Taxonomy (What is the current name?) – Biostatus (Is this organism here? Exotic or native?) – Associations (What plant is it found on?) – Collection specimen data • Relevant for informing import and export policy and knowing what is in the environment • http://NZfungi2.LandcareResearch.co.nz/ • Feeds through to NZOR – Jerry Cooper Link seminar 26th November 2013 NZfungi database NZfungi database Armillaria novae-zealandiae Management of Psa?

• Psa can be controlled by antibiotics – improper use problem – Resistance problem – EU zero residue problem – Problems can be mitigated • Need a novel biological solution: – KVH Investigating biologicals – Screened ICMP collection for anti-Psa bacteriocins – Found 10 promising candidates (from 200) Management of Psa? Conclusions

• Pathogens don't carry passports • Need taxonomic input to guide policy development – Identification & interpretation can be very complicated • Species / pathovars / populations / genes • Preparedness for emerging pathogens • Myrtle rust taxonomy uncertain • Taxonomy changing rapidly, need to stay up to date with what we have here • Need taxonomic input to guide policy development

Conclusions

• LCR can help with future issues – Collections – Databases – Staff capability

Acknowledgements

• LCR technical staff – Maureen Fletcher, Paula Wilkie, Duckchul Park • MPI PHEL Tamaki – Brett Alexander, Rob Taylor • Funding – LCR, KVH, MPI